Phylogeny of Campanulaceae S. Str. Inferred from Its Sequences of Nuclear Ribosomal DNA

W. M. M. Eddie; T. Shulkina; J. Gaskin; R. C. Haberle; R. K. Jansen

The Campanula pyramidalis complex is a group of closely related taxa with a distribution across the Balkans, from the Gulf of Trieste in the north to the Peloponnese Peninsula in the south, with small disjunct parts of the range in the south Apennines. Although 21 taxa were described within this complex, only three, C. pyramidalis, C. versicolor, and C. secundiflora, have been generally accepted in recent synoptical taxonomic treatments. Our molecular phylogenetic analyses based on sequences of three non-coding chloroplast regions (psbA-trnH, psbZ-trnfM, trnG-trnS) as well as of nuclear ribosomal internal transcribed spacers (nrITS), lend strong support to the recognition of several lineages which only partially correspond to generally accepted taxonomic concepts. Molecular data presented in this study showed that C. pyramidalis is a polyphyletic assemblage that segregates into three distinct lineages, one of which is described here as a new species, C. austroadriatica sp. nov. The lectotype of C. pyramidalis, redefined in a strict sense, is designated. Neither C. versicolor nor C. secundiflora were found to be strictly monophyletic, but their monophyly could not be rejected. Morphological and biogeographical implications are discussed.

The circumscription and infrageneric classification of Campanula is highly controversial. Independent and combined data from nuclear and chloroplast sequences (trnL-trnF, ITS) were analyzed with Bayesian and parsimony methods to elucidate the phylogenetic relationships of Campanula and allied genera, and explore the biological processes that occurred during the evolution of this genus. The main sections and subgenera of Campanula and related genera were sampled extensively. Chromosome numbers, corolla types, habit and capsule dehiscence were mapped on the trees to search for evolutionary patterns. The phylogenetic analyses revealed that Campanula, as currently circumscribed, is not monophyletic. This genus is divided into two main clades: a large core of Campanula species that includes related genera (Adenophora, Asyneuma, Azorina, Campanulastrum, Diosphaera, Edraianthus, Githopsis, Hanatnisaya, Heterocodon, Legousia, Michauxia, Petromarula, Physoplexis, Phyteuma, Trachelium, and Triodanis), and a clade constituted by Musschia plus two Campanula species. The large core of Campanula is divided into two main groups, a rapunculoid and a campanuloid group. Both Bayesian and Parsimony analyses indicate that the main morphological characters used in classifications, such as flower shape and capsule dehiscence, have arisen in parallel. Strong selective pressures from pollinators are suggested to explain floral convergence. We put forward two different proposals in order to accomplish a classification of Campanula that more accurately reflects the evolution of this genus.

PHYLOGENY OF CAMPANULACEAE S. STR. INFERRED FROM ITS SEQUENCES OF NUCLEAR RIBOSOMAL DNA 1 W. M. M. Eddie, 2,5 T. Shulkina, 3 J. Gaskin, 3,4 R. C. Haberle, 2 and R. K. Jansen 2 ABSTRACT Ninety-three taxa comprising thirty-two genera (plus four outgroups from Lobeliaceae) were used to estimate a phylogeny of the Campanulaceae based on ITS sequences of nuclear ribosomal DNA. From 2629 most parsimonious trees, a strict consensus tree with bootstrap values was constructed, in addition to a phylogram showing branch lengths. The topologies of these two trees are discussed in relation to the pollen and capsule morphology within the family, in addition to chromosome number and geographical distribution. The results show that there is a major dichotomy between the colpate/colporate pollen alliance (platycodonoid taxa) and the porate pollen alliance (wahlenbergioid and campanuloid taxa). Both these major alliances are monophyletic. Within the porate alliance there are two major clades, the wahlenbergioids and the campanuloids. The campanuloid clade is further subdivided into two major clades representing the Rapunculus and the Campanula s. str. groups of taxa, plus three smaller clades that are considered as ‘‘transitional’’ taxa. It is argued that the family originated in a fragmenting West Gondwanaland and that tectonic processes are responsible for the major dichotomy in the family. The colpate/colporate platycodonoids subsequently remained relatively relictual in Asia, whereas the porate taxa spread over much of the Northern and Southern Hemispheres. The campanuloid lineage spread over the Northern Hemisphere from a major evolutionary center in the Mediterranean region and is represented in North America only by the Rapunculus group. The wahlenbergioid lineage is widely dispersed across the southern continents and oceanic islands but has a major secondary center of diversification in southern Africa. The use of ITS provides insights for future investigations and a phylogenetic framework that can be tested with other data sets. Its limitations for phylogeny reconstruction are briefly discussed. More extensive taxon sampling and additional data sets are required to refine these results and for a new classification of the Campanulaceae to be proposed. Key words: Campanulaceae, evolution, Gondwanaland, ITS, nuclear-ribosomal DNA, phylogeny. Classification systems of the bellflower family (Campanulaceae s. str.) have traditionally followed the arrangements of Boissier (1875, 1888) and Schönland (1889–1894) and, together with the refinements of Charadze (1949, 1970, 1976), Fedorov (1957), and others, can ultimately be traced back to the arrangement of De Candolle (1830) who di- vided the family into two subtribes, the Campanuleae and the Wahlenbergeae, based on the mode of capsule dehiscence (Table 1). Schönland divided the family into three subtribes, separating Platycodon A. DC., Musschia Dum., and Microcodon A. DC. in his subtribe Platycodinae on the basis of calyx lobe position in relation to the locules of the 1 W.M.M.E. thanks Tina Ayers and Randy Scott for their hospitality in Flagstaff and for facilities at Northern Arizona University in 1998. Others who deserve special mention include Ian Oliver, Andrew Hudson, Ian Hedge, Martin Ingrouille, Susana Neves, Mark Chase, Mike Fay, Peter Lewis, Marcia Ricci, Jim and Jenny Archibald, and Per Hartvig. For technical support we thank Kavita Vyas and Christine Green. The Regius Keepers of the Royal Botanic Garden, Edinburgh, and the Directors of the Royal Botanic Gardens, Kew, are thanked for use of facilities. Assistance by the staff of the Goulandris Natural History Museum (Athens), the Royal Botanic Garden, Edinburgh, and the Darwin Library (University of Edinburgh) is gratefully acknowledged. For funding, W.M.M.E. acknowledges The University of London (The Central Research Fund; The Keddy Fletcher-Warr Studentship of Birkbeck College), The University of Edinburgh (The Molecular Biology Fund of the Institute of Cell and Molecular Biology; The James Rennie Bequest), and The Royal Botanic Garden, Edinburgh (The Edinburgh Botanic Garden (Sibbald) Trust). R.C.H. acknowledges assistance with collections from and discussions with Nancy Morin (The Arboretum at Flagstaff) and Barbara Ertter (Jepson Herbarium, University of California, Berkeley). We also thank Tom Givnish and Tom Lammers for helpful suggestions on an earlier version of the manuscript. This research was supported by NSF grant DEB 9982092 to R.K.J. 2 Section of Integrative Biology, Institute of Cellular and Molecular Biology, and Plant Resources Center, University of Texas at Austin, Austin, Texas 78712, U.S.A. jansen@mail.utexas.edu. 3 Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, U.S.A. 4 Present address: United States Department of Agriculture, Agricultural Research Service, Northern Plains Agricultural Research Laboratory, 1500 North Central Avenue, Sidney, Montana 59270, U.S.A. 5 Present address: Office of Lifelong Learning, University of Edinburgh, 11 Buccleuch Place, Edinburgh, Scotland, U.K. weddie1@staffmail.ed.ac.uk ANN. MISSOURI BOT. GARD. 90: 554–575. 2003. Volume 90, Number 4 2003 Table 1. Eddie et al. Phylogeny of Campanulaceae Classification of Campanulaceae (A. P. de Candolle, 1830). Subtribe I (Wahlenbergeae) Subtribe II (Campanuleae) Capsule with apical (valvate) dehiscence Campanumoea Blume (baccate capsule) Canarina L. (baccate capsule) Cephalostigma A. DC. Codonopsis Wall. Jasione L. Lightfootia L’Her. Microcodon A. DC Platycodon A. DC. Prismatocarpus L’Her. Roella L. Wahlenbergia W. Roth Capsule with lateral (porate) dehiscence Adenophora Fisch. Campanula L. Merciera A. DC. (indehiscent) Michauxia L’Her. Musschia Dum. Petromarula Vent. ex Hedw. f. Phyteuma L. Specularia A. DC. Symphyandra A. DC. Trachelium L. ovary (Table 2). Such natural classifications were essentially based on morphology of the calyx (e.g., the presence or absence of appendages between the lobes) or of the mode of capsule dehiscence (e.g., whether it is apical and valvate or lateral and porate). Many authors (e.g., Hutchinson, 1969; Carolin, 1978; Cronquist, 1988; Takhtajan, 1969) considered Cyananthus A. DC. to be the most primitive genus within the family based on its superior ovary. These various classifications were generally useful in floristic works, especially during the 20th century when much of the research on the Campanulaceae was of a regional, floristic nature. Frequently, various authors have used their own modified system with many nomenclatural changes, and great confusion has resulted. Considerable conflict still exists as to the number of genera recognized. Table 2. 555 Generic distinctions in the family are often subtle, being based on a suite of characters best observed in living plants. In addition, species of the Campanulaceae appear to be prone to considerable phenotypic plasticity (Eddie, 1997; Eddie & Ingrouille, 1999) as well as ontogenetic variation, and this has led to a burgeoning of the literature with superfluous species names. The few generic monographs that have been completed, although excellent, often lacked a global perspective, and have contributed little to the establishment of a new, more generally accepted classification of the family. Reconstruction of the phylogeny of the Campanulaceae has been hindered by a lack of consensus as to what constitutes a genus and the failure to apply important character combinations (e.g., cytological and palynological characters), which could potentially Classification of the Campanulaceae (Schönland, 1889–1894). Tribe Campanuleae Subtribe Campanulinae Subtribe Wahlenberginae Subtribe Platycodinae Adenophora Fisch. Campanula L. sect. Medium Tourn. sect. Rapunculus Boiss. Canarina L. Heterocodon Nuttall Michauxia L’Her. Ostrowskia Regel Peracarpa J.D. Hooker & T. Thoms. Phyteuma L. sect. Cylindrocarpa Rgl. sect. Hedranthum G. Don sect. Petromarula A. DC. sect. Podanthum G. Don sect. Synotoma G. Don Symphyandra A. DC. Trachelium L. Campanumoea Blume Cephalostigma A. DC. Codonopsis Wall. Cyananthus Wall. Githopsis Nuttall Hedraeanthus Grisebach Heterochaenia A. DC. Jasione L. Leptocodon (J. D. Hooker) Lem. Lightfootia L’Her. Merciera A. DC. Prismatocarpus L’Her. Rhigiophyllum Hochst. Roella L. Siphocodon Treichelia Wahlenbergia W. Roth Microdon A. DC. sect. Eumicrocodon A. DC. sect. Caelotheca A. DC. Musschia Dum. Platycodon A. DC. 556 Annals of the Missouri Botanical Garden highlight major discontinuities at the generic, tribal, and subtribal levels. Many species have been placed, for convenience, in Campanula L., Asyneuma Grisebach & Schenk, and Wahlenbergia Schrad. ex W. Roth, and this has further complicated our understanding of phylogenetic relationships. Indeed, some of the intrageneric taxa in these large genera are probably more deserving of generic status than some of the currently recognized segregate genera. The so-called satellite genera of Campanula do not appear to be any closer to each other than they do to Campanula, and there is no evidence to suggest that Campanula, despite its numerical superiority, is ancestral to them. It is thus often easier to define what Campanula is not rather than what its actual boundaries are. Thus, to some extent, the genus Campanula is conceptually useless and its continued use as a ‘‘core’’ genus may be misleading. The same is probably true for Asyneuma and Wahlenbergia. Knowledge of inter- and intrageneric relationships within the family has steadily increased during the latter half of the 20th century. Cytological studies, beginning with the seminal investigations of Gadella (1962, 1963, 1964, 1966, 1967), Contandriopoulos (1964, 1966, 1970, 1971, 1972, 1976, 1980a, b, 1984), Contandriopoulos et al. (1972, 1974, 1984), Damboldt (1965a, b, 1966, 1968, 1969, 1970, 1975, 1976, 1978a, b), Phitos (1963a, b, 1964a, b, 1965), and Podlech and Damboldt (1964) have vastly increased our knowledge of intrageneric relationships, particularly of the genus Campanula. The most common chromosome number in the Campanulaceae is n 5 17, and this appears to have evolved independently several times in relatively unrelated genera (e.g., in Campanula, Nesocodon M. Thulin, Ostrowskia Regel, and Canarina L.). Forty-two percent of the published chromosome counts of the family Campanulaceae s.l. have this number (Lammers, 1992). The base number in the family has been suggested to be x 5 8 (Böcher, 1964; Contandriopoulos, 1984), but Raven (1975) suggested that x 5 7 is the ancestral number. An ancestral base number of x 5 7 is supported by counts for Cyananthus (Kumar & Chauhan, 1975; Hong & Ma, 1991). It was Avetisian (1948, 1967, 1973, 1986) who first drew attention to the different pollen morphologies within the family and gave a schematic presentation of pollen evolution based on aperture types. She further pointed out that pollen with colpate and colporate apertures are typical of those taxa found in the tropics, whereas those with porate apertures are typical of taxa from temperate regions. Dunbar (1973a, b, c, 1975a, b, 1981, 1984) and Dunbar and Wallentinus (1976) extended Avetisian’s work by providing excellent surveys of pollen from numerous genera of the Campanulaceae, and this has been augmented by Morin (1987), Nowicke et al. (1992), and Morris and Lammers (1997). Several of these studies suggest that some of the genera are artificially grouped together in De Candolle’s and in Schönland’s arrangements because of the limited criteria used as the basis for their classification systems. Seed morphology has been examined for a number of taxa, principally those of North America (Shetler & Morin, 1982, 1986) and Eurasia (Belyaev, 1984a, b, 1985, 1986; Oganesian, 1985). Life-form in the Campanulaceae has been studied intensively by Shulkina (1974, 1975a, b, 1977, 1978, 1979, 1980a, b, c, 1986a, b, 1988) and Shulkina and Zykov (1980), but these data have not been incorporated into a cladistic analysis. Serological studies have been done on the tribe Phyteumatae (Gudkova & Borshchenko, 1991), while Gorovoi et al. (1971) conducted a limited chemotaxonomic survey of Russian Far-Eastern taxa. Kolakovsky (1980, 1982, 1986a, 1986b, 1987), Kolakovsky and Serdyukova (1980), and Lakoba (1986) did some pioneering carpological investigations of the family, but so far this work has not been corroborated and it remains to be seen whether their segregate genera will be accepted. Few molecular phylogenetic studies of the Campanulaceae have been undertaken. Cosner (1993) and Cosner et al. (2004) used chloroplast DNA (cpDNA) structural rearrangements to establish a phylogeny of the family based on 18 genera, while Cosner et al. (1994) determined rbcL sequences for several genera as part of a study of interfamilial relationships of the Campanulales. Eddie (1997, and unpublished data), using cladistic and phenetic methodologies, investigated the morphology of most of the genera of the Campanulaceae, in addition to molecular variation of 23 to 29 taxa using internal transcribed spacers (ITS) and matK/trnK-intron sequence data from nuclear ribosomal (nrDNA) and cpDNA, respectively. For molecular variation within and between genera, ITS sequences have been used by Ge et al. (1997) for Adenophora Fisch. and by Kim et al. (1999) for Hanabusaya Nakai. Haberle (1998) examined relationships among the families Campanulaceae, Cyphiaceae, Nemacladaceae, Cyphocarpaceae, and Lobeliaceae using ITS sequence data. This study is an attempt to reconstruct the phylogeny of the Campanulaceae s. str. using nrDNA ITS sequences and to compare the results with certain characters that have traditionally been used in Volume 90, Number 4 2003 Eddie et al. Phylogeny of Campanulaceae the classification of the Campanulaceae (i.e., capsule morphology and presence/absence of calyx appendages in addition to chromosome numbers, pollen, and geographical distribution). It is the first time that molecular methods have been applied to a broad sample of taxa (93 species in 32 genera) within the family. This study is also the first part of more extensive investigations of the Campanulaceae using a variety of molecular markers, including the sequences of chloroplast genes matK and rbcL, as well as chloroplast genome rearrangements and morphological data. Ultimately these studies should lead to a new comprehensive classification of the Campanulaceae. modifications such as the addition of PVP-40 and/ or BSA. Double-stranded DNA from the ITS and the intervening 5.8S subunit of the 18S–26S nr DNA was amplified using standard PCR procedures (Kim & Jansen, 1994). The basic primer sequences were those of White et al. (1990) or the modifications by Yokota et al. (1989). Purification of the PCR products was by means of Qiagen QIAquick spin columns (Qiagen Corp.), and sequences were obtained from ABI Prism 377 Automatic DNA Sequencers (Perkin Elmer, Applied Biosystems Division). For each taxon, forward and reverse sequences were obtained, and the results were saved as electropherograms and edited using the programs SEQUENCHER, vers. 3.0 and 4.1.2 (Gene Codes Corp.), EDITVIEW, ver. 1.0.1, and SEQUENCE NAVIGATOR, ver. 1.0.1 (Perkin Elmer, Applied Biosystems Division). MATERIALS AND METHODS TAXA SAMPLED AND SOURCES OF PLANT MATERIAL ITS sequences for 93 taxa of the Campanulaceae were used, including a number of which were previously published and available from Genbank (Fu et al., 1999; Ge et al., 1997; Kim et al., 1999; Schultheis, 2001; K. Dotti, unpublished data) (see Appendix 1). Many of the samples represent taxa that are commonly accepted as genera or sections within genera because of their obvious morphological discontinuities and that provide a broad sample of the family. The nomenclature used in this study is based on the classification system used by Fedorov (1957), but the names given to the major groups or clades are merely for convenience and not based on any particular classification system. Added to the data set were four outgroups from the Lobeliaceae (Downingia bacigalupii Weiler, Lobelia aberdarica R. E. Fries & T. C. E. Fries, L. tenera Kunth, and L. tupa L.), bringing the total number of taxa in the data set to 97. There is overwhelming evidence from both morphological (Lammers, 1992; Gustafsson & Bremer, 1995) and molecular (Cosner et al., 1994; Gustafsson et al., 1996; Jansen & Kim, 1996; Albach et al., 2001) studies that the Lobeliaceae are an appropriate outgroup for the Campanulaceae sensu stricto. DNA samples were obtained from living plants cultivated at The Institute of Cell and Molecular Biology (ICMB), University of Edinburgh, Scotland, U.K., The Royal Botanic Garden Edinburgh (RBGE), Scotland, U.K., The University of Texas at Austin (UT), U.S.A., and the Missouri Botanical Garden (MO), St. Louis, U.S.A. For sources of material and location of vouchers, see Appendix 1. DNA ISOLATION, AMPLIFICATION, AND SEQUENCING Genomic DNA was extracted following the CTAB protocol of Doyle and Doyle (1987) or with minor 557 SEQUENCE ALIGNMENT The boundaries for the ITS region were determined by comparisons with published ITS sequences of Nicotiana rustica L. (Solanaceae, Venkateswarlu & Nazar, 1991), Krigia Schreb. (Asteraceae, Kim & Jansen, 1994), Madiinae Benth. (Asteraceae, Baldwin, 1992), and Gentiana L. (Gentianaceae, Yuan et al., 1996). Alignment of ITS proved to be problematic, particularly at the 39 end of the ITS2 region close to the 26S subunit. Due to a high level of ambiguity, this region was deleted at 205 bases downstream from the start of the ITS2 region. The highly conserved 5.8 subunit was not available for all taxa and therefore was not included in phylogenetic analyses. The multiple alignment used in this study was created by CLUSTALX (ver. 1.64b; Thompson et al., 1997) in several stages using the Slow/Accurate dynamic programming option. Divergent sequences (. 40%) were delayed in the alignment procedure. Insertions from individual taxa, which created gaps and had no apparent homology with the rest of the taxa, were removed, and another round of alignment was initiated. A range of gap penalties from 5.00 to 20.00 and gap extension penalties from 3.00 to 8.00 were initially tried with various combinations until a consistent alignment was established using a gap penalty of 8.00 and a gap extension penalty of 5.00. Minor final adjustments to the alignment were done manually. The alignment is available at: ,http://www.biosci. utexas.edu/IB/faculty/jansen/lab/personnel/eddie. htm.. All new sequences have been submitted to Genbank. 558 Annals of the Missouri Botanical Garden Table 3. Base composition and nucleotide divergence in the aligned partial sequences of ITS1 and ITS2 regions of nr DNA in the Campanulaceae. Sequence parameter Aligned length Constant sites Variable sites Informative sites G 1 C content (%) Unambig. transitions Unambig. transversions Ts/Tv ratio Avg. base frequencies* ITS1 1 partial ITS2 A 5 20.8 497 81 416 (75 uninformative) 345 59.8 627 500 1.254 C 5 30.6 G 5 29.2 T 5 19.3 * Missing data and gaps excluded. PHYLOGENETIC ANALYSES A search for the most parsimonious tree was initiated using the PARSIMONY option of PAUP 4.068 (Swofford, 2001) with ACCTRAN, MULTREES, TBR, and COLLAPSE ZERO LENGTH BRANCHES options. All characters were given equal weight and were unordered. Gaps were treated as missing data. The HEURISTIC search algorithm was chosen, with 1000 random addition replicates and with a limit of 2000 trees saved per replicate. The amount of support for monophyletic groups was evaluated by 1000 bootstrap replicates and a 50% cut-off value for the bootstrap consensus tree (Felsenstein, 1985). Consistency Indices (CI) (Kluge & Farris, 1969) were also computed. The Retention Index (RI) and the g1 statistic (Hillis & Huelsenbeck, 1992) were also computed, the latter after computing the tree-length distribution of 100,000 random parsimony trees by means of the RANDOM TREES command. RESULTS AND DISCUSSION The total aligned length of the ITS1 and partial ITS2 (including gaps) was 497 bp. There were 81 constant characters, 71 variable characters that were parsimoniously uninformative, and 345 parsimoniously informative characters (Table 3). Parsimony analyses generated 2629 trees with 2130 steps, a CI of 0.3703 (excluding uninformative characters), and RI of 0.7583 (Figs. 1, 2). The g1 statistic for 100,000 trees randomly sampled was 20.327694 indicating that the ITS data set is significantly skewed from random and contains considerable phylogenetic information (Hillis & Huelsenbeck, 1992). For other statistics of the aligned sequences see Table 3. Multiple ITS types were not detected, and in one case there were two separate samples of the same species (Adenophora divaricata Franch. & Sav.) that did not come out together. The branch lengths are very short for the Adenophora clade overall, which indicates that most of the species have very similar ITS sequences. The differences between the two samples of A. divaricata suggest either misidentification of the original sample or population differences in the ITS sequences. The taxonomic categories used in classifications are unequivocal and the amount of molecular divergence (and hence phylogenetic signal) within and between taxa at each level in the taxonomic hierarchy varies. For a family such as the Campanulaceae, which has numerous monophyletic genera and sections, the use of ITS sequences is justified by the phylogenetic signal obtained, but there may be substantial trade-off due to problems with alignments. The difficulties associated with sampling across a wide spectrum of taxa in the Campanulaceae should lessen as we are able to refine our molecular analyses at different levels in the taxonomic hierarchy, in conjunction with other sources of data. Due to high ambiguity at the generic level in the Campanulaceae, ITS sequence data may be approaching the limits of usefulness for phylogenetic reconstruction, whereas at the species level, there may not be enough signal, and many species may be spuriously placed with each other. For extensive discussion of the utility and limitations of the ITS region in the reconstruction of angiosperm phylogeny, see Baldwin et al. (1995), Coleman (2003), and Goertzen et al. (2003). MAJOR CLADES IN THE ITS TREE The topology of the strict consensus tree (Fig. 1) shows that there are two major clades of the Campanulaceae. This major dichotomy in the family is supported by pollen data. For convenience, these two major clades are referred to as alliances and are named on the basis of their pollen types. The taxa in the smaller of these two alliances comprise Volume 90, Number 4 2003 Eddie et al. Phylogeny of Campanulaceae 559 Figure 1. Strict consensus of 2629 most parsimonious trees with 2130 steps for 93 taxa of the Campanulaceae and 4 outgroups of the Lobeliaceae based on parsimony analysis of the combined ITS1 and ITS2 sequence data. The numbers above the nodes are bootstrap percentages of 1000 replicates. [CI 5 0.3703 (excluding uninformative characters), RI 5 0.7583.] Nodes without bootstrap values had less than 50% support. 560 Annals of the Missouri Botanical Garden Figure 2. Phylogram of one of the 2629 equally parsimonious trees for 93 taxa of the Campanulaceae and 4 outgroups of the Lobeliaceae based on parsimony analysis of the combined ITS1 and partial ITS2 sequence data. A scale bar representing 10 changes is shown on bottom left corner. Volume 90, Number 4 2003 Eddie et al. Phylogeny of Campanulaceae 561 genera such as Codonopsis, Platycodon, Canarina, etc., which are all distinguished by their possession of either colpate or colporate pollen (Avetisian, 1948, 1967, 1973, 1986; Dunbar, 1973a, b, c, 1975a, b, 1981, 1984) and are also referred to as the platycodonoid group in this paper. The colpate/ colporate alliance is strongly supported with a 100% bootstrap value and is the only clade with taxa that have baccate fruits (Canarina, Campanumoea Blume, and Cyclocodon W. Griff.), although the majority have dry capsules. Geographically, the colpate/colporate alliance is mostly distributed in the tropics or subtropics, from Southeast Asia and the western Himalayas to Ussuriland, Korea, and Japan, and from Indonesia and the Philippines to New Guinea. The genus Canarina is unique within this alliance in being essentially African, but it is disjunct, with one species in Macaronesia and two species in the mountains of East Africa. The taxa in the larger alliance comprise the remainder of the Campanulaceae, and they are distinguished by their porate pollen. The porate alliance has only weak support with a 55% bootstrap value. It is far larger numerically than the colpate/colporate alliance and is divided into two major groups, the wahlenbergioids and the campanuloids. This huge alliance is distributed mostly in the temperate regions of the world, although a few wahlenbergioid and campanuloid taxa extend to the tropics. All taxa within these two groups have capsules that are predominantly dry and dehiscent. In the discussion that follows, we describe the major groups in the two alliances and how they compare with data from morphology, chromosome number, and geography. tains of southern Asia. Because of its superior ovary and low chromosome number of 2n 5 14, it has traditionally been considered the most ancestral genus of the Campanulaceae (Hutchinson, 1969; Cronquist, 1988; Takhtajan, 1969). However, it also has specialized ecological characters such as deep taproots and prostrate lateral branching, both of which are characteristic of alpine plants. The isolated position of Canarina is supported by both geography and chromosome number. Canarina canariensis (L.) Vatke has 2n 5 34, while the remainder of the platycodonoids have 2n 5 16 or 18. Bootstrap support for the clade comprising Leptocodon, the remainder of Codonopsis, plus Campanumoea javanica and Platycodon is moderate (74%). Support for the minor clade containing the bulk of Codonopsis plus Platycodon and C. javanica is strong (88%), but the clade with only the latter two genera is moderately supported (70%). The taxa of Codonopsis are morphologically less divergent from each other, whereas C. javanica and Platycodon are considerably divergent. Hong and Pan (1998), on the basis of pollen morphology, seed coat, and gross morphology, restored the genus Cyclocodon, which was formerly included in Campanumoea s.l. as C. celebica Blume and C. lancifolia (Roxb.) Merr. They considered Cyclocodon to be more closely related to Platycodon than to Campanumoea s. str. (i.e., C. javanica Blume and C. inflata (Hook. f.) C. B. Clarke). Campanumoea and Cyclocodon have baccate fruits but would appear to be rather distant from Canarina. THE PORATE ALLIANCE (THE WAHLENBERGIOID AND CAMPANULOID GROUPS) THE COLPATE/COLPORATE ALLIANCE (THE PLATYCODONOID GROUP) There is strong support (100%) for the monophyly of the colpate/colporate alliance, although the major clades within this alliance are only partially resolved. Canarina, Cyananthus, and Codonopsis Wall. subg. Obconicapsula D. Y. Hong form a polytomy with the remainder of taxa, including Codonopsis, Leptocodon (J. D. Hooker) Lem., Platycodon, and Campanumoea javanica Blume. Codonopsis subg. Obconicapsula is somewhat isolated morphologically and, to a lesser extent, geographically (central Himalayas) from the rest of Codonopsis. It has an ovary that bulges upward above the level of the calyx lobes and an incomplete nectar dome. These features, together with the overall appearance of the flower, recall Platycodon. Cyananthus comprises highly adapted perennial and annual species of very high elevations in the moun- The monophyly of the porate alliance is weakly supported (55%) and it comprises two very unequal clades, the wahlenbergioids and the campanuloids. This is undoubtedly an artifact of the undersampling of wahlenbergioid taxa. The wahlenbergioid group. The sister relationship of the two wahlenbergioid taxa, Craterocapsa Hilliard & B. L. Burtt and Roella L., has strong bootstrap support (94%). These two genera have traditionally been considered closely related (Hilliard & Burtt, 1973). Both are from southern Africa, although Craterocapsa ranges north to the mountains of eastern Zimbabwe. Since only three traditionally accepted wahlenbergioid genera were available for molecular analysis, the discussion of the results for this group is relatively straightforward, but caution should be observed for such a small sample. Wahlenbergia hederacea L. falls within the campanuloid group and is therefore distant from the other two 562 Annals of the Missouri Botanical Garden wahlenbergioid genera. This is surprising because this species has traditionally been considered as typically wahlenbergioid. It has a chromosome number of 2n 5 36, which is not particularly unusual, but it is isolated in western Europe, and has a vegetative morphology that is rather atypical for the wahlenbergioids. Although all modern European workers have never questioned the wahlenbergioid nature of W. hederacea, this species was recognized as a separate genus by some early workers (Schultesia Roth, Valvinterlobus Dulac, Aikinia Salisb. ex A. DC.) and it was assigned to Roucela by Dumortier (1827). The majority of species of Wahlenbergia are distributed in the Southern Hemisphere. Some species (e.g., W. trichogyna Stearn) have 2n 5 36, but the majority have 2n 5 18 (see also Petterson et al., 1995; Crawford et al., 1994; Anderson et al., 2000). In the study of Cosner et al. (2004), the Australian species, W. gloriosa Lothian (not sampled), was found to be in the same clade as Roella ciliata L. Damboldt] erinus L., Campanula mollis L., and Campanula edulis Forssk.), but Azorina vidalii (Wats.) Feer, with its nodding flowers, is a conspicuous exception. With Trachelium removed, bootstrap support for this clade is 93%. Campanula (subg. Roucela) erinus (2n 5 28) belongs to a rather distinct group of annual campanuloids of the Mediterranean, which superficially resemble C. mollis and C. edulis, but its capsules are discoid and the calyx appendages are absent. The corolla approaches the hypercrateriform shape of Trachelium corollas to some extent. The flowers of Diosphaera Buser are quite similar to those of Trachelium and it has the same chromosome number (2n 5 34), but there are conspicuous differences between the two genera, both vegetatively and in the form of the inflorescence. The two genera are often united, but they are disjunct geographically in the Mediterranean. Calyx appendages are absent in both genera. Azorina Feer is quite isolated morphologically (vegetatively and in branching pattern), but its vague resemblance to Campanula bravensis Bolle and C. jacobaea C. Smith of the Cape Verde Islands, together with its chromosome number of 2n 5 56, may link it rather tenuously to Campanula subsect. Oreocodon Fed. (but see also Thulin, 1976: 354). Support for the clade that comprises Azorina, Feeria, Campanula mollis, and C. edulis is weak (58%), but when Azorina is removed support for the remaining taxa is 100%. Feeria angustifolia has traditionally been associated with Trachelium, but morphologically it is quite distinct. In some respects, particularly the globular, more compact shape of the inflorescence, and the valvate apical dehiscence, it approaches Jasione L., but the chromosome number for Feeria angustifolia is 2n 5 34 (vs. 2n 5 12 for Jasione). The similarity of its ITS sequences with those of both Campanula mollis and C. edulis does not accord with its morphology. Campanula mollis and C. edulis are probably closely related to each other, and this relationship is strongly supported in the ITS tree (96%). These two species belong to a group of annual and perennial campanuloids (2n 5 24, 28, 54, 56), which range from Macaronesia, North Africa, and the Iberian Peninsula south to the equator in Tanzania. They have basal dehiscence and appendages between the calyx lobes (Maire, 1929; Quézel, 1953; Thulin, 1976). This group probably links up with Campanula subsect. Oreocodon of the western Himalayas and south-central Asia, which is characterized by species such as C. incanescens Boiss., C. cashmeriana Royle, and C. colorata Wall. The remaining taxa in the Campanula s. str. clade are weakly supported (58%) as a monophy- The campanuloid group (Campanula s. str., ‘‘transitional’’ taxa, and Rapunculus clades). This huge group forms an unresolved polytomy consisting of two major clades and three smaller ones. This basic division is partially in agreement with mode of capsule dehiscence (there are exceptions such as Edraianthus in the Campanula s. str. clade and Adenophora and subsection Heterophylla in the Rapunculus clade) and presence or absence of calyx appendages, two characters that have traditionally been used in intrageneric classifications of Campanula (Boissier, 1875, 1888; Fedorov, 1957). One large, well-supported clade (81%) comprises those taxa centered around Campanula s. str. (i.e., mostly those taxa belonging to the sect. Medium DC.), but also genera such as Trachelium, Diosphaera, Azorina, etc. The second large clade has moderate support (69%) and comprises those taxa centered around Campanula sect. Rapunculus (Fourr.) Boiss. (the Rapunculus clade). Two smaller clades have strong support (100%) and consist of several transitional genera such as Jasione L., Musschia, and Gadellia Shulkina, while the third small clade comprises Wahlenbergia hederacea alone. THE CAMPANULA S. STR. CLADE The Campanula s. str. clade includes a small number of mostly monotypic genera that are considerably more divergent than the majority of taxa in this clade. Some have upright flowers (e.g., Trachelium caeruleum L., Diosphaera rumeliana (Hampe) Bornm., Feeria angustifolia (Schousb.) Buser, Campanula [subg. Roucela (Dumort.) J. Volume 90, Number 4 2003 Eddie et al. Phylogeny of Campanulaceae letic group. They are mostly Eurasian and North African, although at least one species in this alliance occurs as far east as the Aleutian Islands (C. chamissonis Fed. subsect. Scapiflorae (Boiss.) Fed., not sampled), and another south to the equator in northern Tanzania (C. keniensis Thulin, also not sampled). The isolated species C. mirabilis Albov (subsect. Spinulosae (Fom.) Fed.) is the sister taxon to all the others. The small clade formed by Edraianthus pumilio (Schultes) A. DC., E. graminifolius (L.) A. DC., and C. latifolia L. is weakly supported (, 50%). The two species of Edraianthus (A. DC.) DC. are confined to the mountains of southeastern Europe, and are rather dissimilar morphologically. Edraianthus pumilio has solitary flowers on multiple inflorescence stems, whereas E. graminifolius has a glomerulate inflorescence. Morphologically, E. pumilio may be closer to Campanula (Petkovia Stefanoff) orphanidea Boiss. (not sampled), which has a similar mode of capsule dehiscence (Hartvig, 1991) and similar corollas (C. orphanidea has 2n 5 26). Edraianthus was formerly considered to be wahlenbergioid because of the apical rupture of its capsule, but its overall morphology is very similar to Campanula and its chromosome number (2n 5 32) is more typical of campanuloid taxa. Campanula latifolia is rather isolated in the Campanula s. str. clade. It belongs to a distinct group of tall mesophytic species from Eurasia that lack appendages and have nodding flowers on long spicate inflorescences (e.g., C. trachelium L., C. bononiensis, C. rapunculoides L., etc.). In general morphology this group (subsect. Eucodon (A. DC.) Fed.) resembles Adenophora. Several other minor groups within the Campanula s. str. clade have moderate to strong support. Michauxia tchihatchewii Fisch. & C. A. Meyer and C. barbata L. have a bootstrap value of 98%. This relationship is surprising since the morphology of these two species is very divergent. The monophyly of the two, yellow-flowered species from the European Alps, C. thyrsoides L. and C. petraea L., is moderately supported (71%). Collectively, these four taxa form a strongly supported clade (85%). The long branches (Fig. 2) show clearly that these four taxa are all very divergent from each other. In some cases, relationships in the Campanula s. str. clade are in accord with classification of Fedorov (1957), whereas in other instances there is conflict. For example, C. armazica Kharadze, C. sosnowskii Kharadze, and C. bellidifolia Adam have a support value of 74%, which agrees with their placement in section Scapiflorae (Boiss.) Fed. In contrast, C. hohenackeri Fisch. & C. A. Mey. (subsect. Triloculares Boiss.) and C. grossheimii Kharadze (sub- sect. Eucodon) have bootstrap support of 100%, but their relationship conflicts with Fedorov’s arrangement. THE ‘‘TRANSITIONAL’’ 563 TAXA The clade comprising Musschia, Gadellia, and the two species of Campanula sect. Pterophyllum Damboldt (C. peregrina L. and C. primulifolia L.) is strongly supported (100%). Musschia aurea Dumort. is an endemic of Madeira together with its congener, M. wollastoni Lowe, whereas C. peregrina and C. primulifolia are disjunct between the eastern Mediterranean region and the western Iberian Peninsula, respectively. Gadellia lactiflora (M. Bieb.) Shulkina is endemic to the Caucasus region. Morphologically, Musschia is different from the other three taxa except for a vague similarity of form, robustness, and disposition of the stigmatic lobes. Its capsule is 5-loculed, prismatic, and opens with numerous transverse slits. Its chromosome number is 2n 5 32. Gadellia was erected by Shulkina (1979) for Campanula lactiflora M. Bieb. based on its distinct growth form and chromosome number (2n 5 36). It has open, upright flowers and dehisces somewhat medially/apically. Campanula primulifolia was placed in the genus Echinocodon (5 Echinocodonia Kolak.) by Kolakovsky (1986b). Campanula peregrina was acknowledged to be very close to C. primulifolia by Damboldt (1978b) and was placed in the section Pterophyllum. Bootstrap support for a close relationship between these two species is 87%. Despite their strong resemblances, the chromosome number for C. primulifolia is 2n 5 36, while C. peregrina is recorded as 2n 5 26 (Gadella, 1964). However, Marchal (1920) recorded the former also as 2n 5 26, so these findings require clarification. The genus Jasione L. is strongly supported as a monophyletic group (100%). Within the genus, J. crispa (Pourr.) Samp. is sister to all the others sampled, but the clade formed by them is weakly supported (64%) and relationships among species within the group are unresolved. The relationship of Jasione to other taxa of Campanulaceae is unresolved in the ITS tree. Jasione has most frequently been associated with the wahlenbergioid alliance, although it does bear some resemblance to Feeria Buser with which it shares a similar mode of capsule dehiscence, but it has a chromosome number of 2n 5 12 (vs. 2n 5 34 for Feeria). THE RAPUNCULUS CLADE The Rapunculus clade has moderate support (69%) and has a number of smaller clades that are 564 Annals of the Missouri Botanical Garden all relatively divergent from each other morphologically. In terms of branch length, the taxa within the Rapunculus clade are much more divergent overall than the taxa within the Campanula s. str. clade (Fig. 2). Githopsis Nuttall and Heterocodon Nuttall are rather divergent in morphology from each other, particularly that of the capsule (see McVaugh, 1945), but are probably closely related and have strong bootstrap support (100%). They are sister to the remaining members of the Rapunculus clade. Most of these taxa are either Mediterranean or North American in distribution. The majority of taxa within this clade have open, upright flowers that are rather stellate in form, and the capsule opens apically or medially by a pore, but there are conspicuous exceptions (see below). None of the taxa in the Rapunculus clade has calyx appendages. The irregular rupture of the capsule apex in Githopsis may represent a derived condition, but this is not to imply that its ancestral state was lateral (e.g., it may be derived from an apical valvate condition similar to that present in the wahlenbergioid alliance). In Adenophora, Hanabusaya, and Campanula rotundifolia L. (the sole representative of the harebell group sampled, Campanula subsect. Heterophylla Fed.), the flowers are campanulate and nodding and the capsule opens basally. The inclusion of these taxa within the Rapunculus clade is surprising. Morphologically these taxa seem to be more closely allied to groups within the Campanula s. str. clade (e.g., C. latifolia and its allies in sect. Eucodon). When Githopsis and Heterocodon are removed, the remaining taxa of the Rapunculus clade have 100% bootstrap support. Within this clade the Texan endemic annual Campanula reverchoni A. Gray is sister to all the remaining taxa, although support for this group is weak (, 50%). Within this clade there are several small groups with moderate to strong support. The clade comprising Adenophora and Hanabusaya is strongly supported (99%), although species relationships are largely unresolved. This confirms the close relationship between Hanabusaya and Adenophora suggested previously by Eddie (1997) and by Kim et al. (1999), and it tentatively suggests that Hanabusaya is closest to the two species A. stenanthina (Ledeb.) Kitagawa and A. paniculata Nannf. (sect. Thyrsanthe (Borb.) Fed.). Support for the clade uniting these three taxa is weak (, 50%). The remaining species of Adenophora form an unresolved polytomy, although there is weak support for a group consisting of A. himalayana Feer (sect. Pachydiscus Fed.) and A. lobophylla D. Y. Hong (sect. Microdiscus Fed.). The sister group to the Adenophora/Hanabusaya clade is only weakly supported, but it contains several well-supported smaller groups. These taxa are divergent morphologically and have a wide range of chromosome numbers. The group containing the serpentine endemic from the Balkans, C. hawkinsiana Hausskn. & Heldreich (2n 5 22), and Iberian endemics C. lusitanica Loefl. (2n 5 18), C. herminii Hoffmanns & Link. (2n 5 32), and C. arvatica Lag. (2n 5 28), is strongly supported (98%), while the clade with C. stevenii M. Bieb. (2n 5 32) and C. persicifolia L. (2n 5 16, 18) has a support value of 99%. The two morphologically divergent species, C. arvatica and C. rotundifolia (2n 5 34), are sister species with 77% bootstrap support. Campanula carpatica Jacq. (subsect. Rotula Fed.) does not appear to be as close to C. pyramidalis L. (2n 5 34), but it does resemble C. herminii from the Iberian Peninsula. Campanula pyramidalis is part of the ‘‘isophylloid’’ group of species (e.g., C. isophylla Moretti, C. garganica Tenore, C. versicolor Andrews [not sampled], etc.), which is centered in Italy and the western Balkan Peninsula and is somewhat intermediate between the Phyteuma L./ Asyneuma alliance and those species that could be considered as typically rapunculoid (e.g., Campanula carpatica, etc.) (see also Damboldt, 1965a). However, many species in this group hybridize freely, and numerous hybrids involving C. carpatica are known in cultivation (Lewis & Lynch, 1989). Thus, the ITS data suggest that this grouping is a natural one. Broader sampling would perhaps have helped clarify the positions of the ‘‘isophylloid’’ and Heterophylla groups. The Phyteuma clade includes morphologically similar species and has strong bootstrap support (91%). Petromarula Vent. ex Hedw. f. is sister to all the other taxa, followed by Asyneuma japonicum (Miq.) Briq. The clade comprising Physoplexis (Endl.) Schur and Phyteuma has a bootstrap support of 81%, but relationships within this group are unresolved. The long branches in this clade (Fig. 2) suggest these taxa are relatively divergent. The sister group of Phyteuma and closely related genera includes Eurasian genera such as Legousia Dur. and several diverse North American taxa, such as Triodanis Raf., Campanula divaricata Michx., and Campanulastrum americanum (L.) Small. This clade is weakly supported with a bootstrap value of less than 50%. Apart from Triodanis, which is sometimes considered to be congeneric with Legousia (McVaugh, 1945, 1948), these taxa are all rather divergent morphologically. In Asyneuma, Phyteuma, Petromarula, Physoplexis, the ‘‘isophylloid’’ species such as Campanula pyramidalis, and the American taxa such as Campanulastrum and Triod- Volume 90, Number 4 2003 Eddie et al. Phylogeny of Campanulaceae anis, the capsule opens apically or medially by a more irregular pore. Morphologically, C. divaricata resembles Adenophora somewhat, and the capsule opens basally. In other respects, such as the open stellate shape and upward orientation of the flower, the majority of the other taxa in this clade are typically rapunculoid (e.g., C. rapunculus L., C. patula L., etc.). The wahlenbergioids probably branched off early in the evolution of the porate alliance and constitute the only major group in the Southern Hemisphere. They have radiated most in southern Africa, although distinctive taxa occur on islands of the Atlantic, Indian, and Pacific Oceans. Several species of Wahlenbergia have ovaries that are almost superior, while Nesocodon from Mauritius has flowers that recall some species of Codonopsis in the colpate/colporate alliance. In contrast, the campanuloids are dominant over much of the Northern Hemisphere. The relative isolation of monotypic or small, distinctive genera within the two main campanuloid clades (e.g., the Rapunculus and Campanula s. str. clades) suggests that the group as a whole evolved in the Mediterranean Basin and spread rapidly over the Northern Hemisphere. The Rapunculus clade is considerably heterogeneous both cytologically and morphologically, although all taxa within this clade are exappendiculate. Many of the species were included in section Rapunculus (Fourr.) Boiss. (Boissier, 1875). It is the most geographically widespread clade, most diverse in the Mediterranean Basin, and the only one that has spread into North America (apart from Campanula chamissonis in the Aleutian Islands). The numerically small but diverse campanulaceous taxa of North America probably contain many relicts from pre-glacial times and represent several relatively independent groups derived from the main rapunculoid radiation in Eurasia (Shetler, 1979). An early radiation of the Rapunculus group in the Northern Hemisphere would explain the distinctiveness of subgroups (e.g., Phyteuma, Petromarula, and related genera) that are associated with the European Alpine orogenic events and fluctuating Mediterranean sea levels during the Tertiary period (Eddie, 1984; Favarger, 1972; Greuter, 1979). It would also explain the presence of endemic genera such as Githopsis in California and the other rather heterogeneous taxa in North America, e.g., Heterocodon and diverse Campanula annuals in California (see Morin, 1980), China, and southern Asia (e.g., Homocodon D. Y. Hong and Peracarpa J. D. Hooker & T. Thoms.). The ancestral group(s) that eventually gave rise to Adenophora, Hanabusaya, and the harebell group (subsect. Heterophylla) may be related to some of the North American taxa such as C. divaricata and C. robinsiae Small (not sampled), and may also have been ancestral to the predominantly appendiculate Campanula s. str. group, of which the mesophytic, exappendiculate species such as C. latifolia, C. trachelium, etc. (sect. Eucodon), may be the least morphologically modified descendants. CONCLUSIONS Overall, there is a remarkable congruence between the ITS tree and traditional ideas on species relationships within the Campanulaceae (Eddie, 1999). The insights of early workers such as De Candolle and Boissier have proved to be remarkably clear, and their classification systems have, on the whole, been logically consistent with our findings on phylogeny. This study also supports the serological studies of Gudkova and Borshchenko (1991) and the cpDNA phylogenies of Cosner (1993) and Cosner et al. (2004). The ITS trees indicate that the colpate/colporate alliance (the platycodonoids) is sister to the remainder of the Campanulaceae (Eddie, 1997, 1999; Shulkina & Gaskin, 1999). This is in agreement with phylogenies of the Campanulaceae based on cpDNA structural rearrangements (Cosner et al., 2004). In comparison with the porate taxa, the colpate/colporate taxa show considerably more molecular divergence, although the wahlenbergioid taxa were under-sampled. As a group, the colpate/colporate alliance has not radiated into drier, more temperate regions and its area of greatest diversity remains the region between the eastern Himalayas and southwest China. It is hypothesized that Ostrowskia (not sampled) represents a minor element of this alliance, which has evolved in the dry, temperate, and highly seasonal environments of Central Asia and thus displays features that parallel certain porate taxa, particularly the mode of capsule dehiscence. Canarina is clearly part of this alliance and was misplaced in the classifications of De Candolle (1830) and Schönland (1889–1894), although its chromosome (2n 5 34) is anomalous within the platycodonoid group. These results also suggest that baccate fruits evolved several times in the colpate/colporate taxa (see Hong & Pan, 1998). Within this alliance there are combinations of certain morphological features that also occur in the porate taxa, e.g., valvate apical dehiscence, a nectary protected by expanded basal filaments (nectar dome), and colored pollen, and these may afford some clues about possible links between the two major alliances of the family. 565 566 Annals of the Missouri Botanical Garden Species of the Campanula s. str. clade are mostly appendiculate, have basal dehiscence, and are cytologically more homogeneous, particularly those species in Campanula and Symphyandra. Many of them were included in Campanula sect. Medium (DC.) Boiss. (Boissier, 1875). Much of the radiation of this group is associated with the mountain-building processes of Eurasia, from the Atlas Mountains in the west to the western Himalayas. Subcenters of high diversity for the Campanula s. str. clade include the Balkan Peninsula, Anatolia, and the Caucasus Mountains. Campanula, as it is currently constituted, is clearly polyphyletic. The more divergent taxa in this clade are found mainly in the Mediterranean basin and are placed in small or monotypic genera (e.g., Azorina, Diosphaera, Edraianthus, Feeria, and Michauxia). Since De Candolle’s monograph of 1830, Edraianthus has been associated with the wahlenbergioid group, but it is clearly campanuloid, although its exact relationships within the Campanula s. str. clade remain unclear (see also Hilliard & Burtt, 1973). Symphyandra A. DC. is now generally considered to be artificial (Greuter et al., 1984; Oganesian, 1995), and this analysis supports that conclusion. However, the four sections of the genus recognized by Fedorov (1957) are all quite distinct, and we suggest that the species formerly included in this genus should be re-examined and not necessarily included in Campanula without substantial evidence. The generic status of Symphyandra odontosepala (Boiss.) E. Esfandiari (not sampled) and the Iranian endemic genus Zeugandra P. H. Davis (not sampled) also need to be reassessed. Symphyandra hofmanni Pant. seems to be rather distant from the bulk of species in Campanula, whereas S. pendula (M. Bieb.) DC. and S. armena (Stev.) A. DC. are much closer. Several genera may best be regarded as transitional between the wahlenbergioid group and the campanuloid group. Musschia is probably better placed with the campanuloids, but it is somewhat intermediate morphologically between the two major porate groups and shows some resemblance to wahlenbergioids such as Heterochaenia A. DC. from Réunion. It does not appear to be close to Platycodon or Microcodon A. DC. as in the arrangement of Schönland (1889–1894). On the basis of ITS sequence similarity to Gadellia, we suggest that the distinct morphological evolution of Musschia on Madeira was relatively rapid. Jasione also appears to be basal within the complex of Northern Hemisphere genera but its exact relationships remain unclear. On the whole it appears to have more affinities with campanuloid taxa. In the cpDNA tree of Cosner et al. (2004), Jasione forms an unresolved polytomy with Symphyandra, Edraianthus, Campanula, and Trachelium. Chromosome numbers (Fig. 2) are lowest overall in the colpate/colporate alliance, although the lowest recorded diploid number is for Jasione (2n 5 12). Within the Rapunculus clade, with the exception of the clade comprising Adenophora and Hanabusaya, the chromosome numbers are diverse and are consistently lower than numbers recorded for the Campanula s. str. clade, which are predominantly 2n 5 34. If we accept the premise that there has been a general increase in chromosome number during the evolution of the Campanulaceae, then the platycodonoids are ancestral to all other groups and the wahlenbergioids and rapunculoids are ancestral to the campanuloids s. str. This accords well with our knowledge of pollen morphology and evolution in the family, as well as the morphology of the capsule in the different groups. However, the diploid number 2n 5 34 occurs in several unrelated lineages (Campanula, Nesocodon, Canarina, and Ostrowskia) and probably evolved independently in each of these genera. This analysis suggests that the ancestral home of the Campanulaceae may be in the region of eastern Asia (of current geography) (see also Hong, 1995; Cosner et al., 2004), but such an interpretation cannot be easily reconciled with the distribution of many genera within the family or with closely related families such as the Lobeliaceae, Cyphiaceae s. str., or Nemacladaceae (Eddie, 1984, 1997, 1999). Carolin (1978), citing the distribution of Cyananthus in India, concluded that the Campanulaceae are essentially an African family that evolved primarily in western Gondwanaland. Bremer and Gustafsson (1997), using nucleotide substitutions in rbcL, suggested an East Gondwanaland origin at the end of the Cretaceous for the asteraceous alliance of families, and that the group subsequently diversified and expanded to West Godwanaland before the breakup of the supercontinent. On the basis of atpB-rbcL spacer sequence data, E. B. Knox (pers. comm.) has stated, ‘‘. . . The interpretation is that Cyphia and the Lobeliaceae originated in southern Africa because the eight ‘basal’ lineages are entirely or predominantly African, and many of these are restricted to southern Africa.’’; ‘‘. . . The Lobeliaceae, Cyphiaceae, and Campanulaceae go back at most 40–50 MYA, and I do not think that the biogeographic patterns can be attributed to Gondwanaland.’’ If the family had arisen in Asia one would have expected platycodonoids to be represented in Eurasia and in North America. The presence of the colporate genus Canarina in Volume 90, Number 4 2003 Eddie et al. Phylogeny of Campanulaceae Africa and Macaronesia suggests that the family may have been more widespread in Africa and around the Indian Ocean than now, but this additional hypothesis does not conflict with an Asian, African, or a Gondwanaland origin for the family. The major dichotomy in the family between the colpate/colporate and the porate taxa suggests that major tectonic processes in the early to mid Tertiary period are implicated in its evolutionary history. A fragmenting West Gondwanaland origin, with the Asian platycodonoid taxa as relictual in land masses that now form the region of the eastern Himalayas and western China, seems a more likely scenario, and this would accord well with the hypothesis (Eddie, 1997) that the more basal members of the wahlenbergioid group are essentially southern or oceanic in their distribution (e.g., Nesocodon, Heterochaenia, Berenice L. R. Tulasne, and the shrubby species of Wahlenbergia from New Zealand, St. Helena, and the Juan Fernandez Islands). The endemic genera of the Cape Region of South Africa probably represent a very early radiation of the wahlenbergioid group in the fynbos vegetation as the climate there cooled and became more arid during the mid to late Tertiary (Eddie & Cupido, 2001). The ITS phylogeny does not necessarily reflect a species phylogeny (Doyle, 1992), but it does provide a basis for inferring possible relationships within and between taxa at several taxonomic levels and provides insights for future investigations. It also provides a phylogenetic framework that can be tested with other data sets. We must await more extensive taxon sampling and data from other genes (both nuclear and chloroplast), as well as intrageneric analyses and chloroplast genome rearrangement studies in order to refine these results. At the same time it must be emphasized that refined data sets of floral morphology and developmental studies are also desirable before a new classification of the Campanulaceae can be proposed. cleaceae, Lobeliaceae, Cyphiaceae) in connection with questions of their systematics and phylogeny. Trudy Bot. Inst. Acad. Sci. Armenia 16: 5–41. [In Russian.] . 1973. Palynology of the Order Campanulales s.l. Pp. 90–93 in L. A. 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Yuan, Y.-M., P. Küpfer & J. J. Doyle. 1996. Infrageneric phylogeny of the genus (Gentianaceae) inferred from nucleotide sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Amer. J. Bot. 83: 641–652. Taxon name and authority Eddie 96086 (UT) Eddie 96087 (UT) Eddie 4548404814 (UT) Cosner s.n. (UT) Gaskin: 463 (UT) Eddie 94003 (UT) Eddie 251.700 (UT) Gaskin: 115 (UT) Cosner s.n. (UT) Haberle (UT) Eddie 96055 (UT) Eddie 95016 (UT) Eddie 98016 (UT) Gaskin 2084 (MO, UT) Eddie 94002 (UT) Neves 227 (UT) Gaskin 205 (UT) RBGE 19875003 Ge et al. (1997) Ge et al. (1997) Ge et al. (1997) Ge et al. (1997) Ge et al. (1997) Ge et al. (1997) Ge et al. (1997) RBGE 19900973 (Japan) Ge et al. (1997) Ge et al. (1997) Ge et al. (1997) Kim et al. (1999) Eddie 4548404814 (EGHB) RBGK 16225 T. Shulkina s.n. (Caucasus, Georgia, MO) NCC 94003 (EGHB) J. Archibald 251.700 (Italy, TEX) Gaskin s.n. (Caucasus, Georgia, TBI) Lammers 8858 (USA, Illinois, F) R. C. Haberle 150 (USA, Virginia, TEX) S. Collenette 8782 (Saudi Arabia, TEX) Eddie 95.016 (Turkey, TEX) Eddie 98016 (EGHB) M. Merello 2084 (Caucasus, Georgia, MO) NCC 94002, Eddie 94002 (EGHB) S. Neves 227 (Portugal, TEX) M. Merello 2194 (Caucasus, Georgia, MO) Genbank accession number (ITS1, ITS2) AY322005, AF090710, AF090716, AF090706, AF090718, AF090714, AF090700, AF090704, AY322006, AF090708, AF090712, AF090702, AF183437, AY322007, AY322008, AY322009, AY322010, AY322011, AY322012, AY322013, AY322014, AY322015, AY322016, AY322017, AY322018, AY322019, AY322020, AY322021, AY331418 AF09071 AF09071 AF09070 AF09071 AF09071 AF09070 AF09070 AY331419 AF09070 AF09071 AF09070 AF18343 AY331420 AY331421 AY331422 AY331423 AY331424 AY331425 AY331426 AY331427 AY331428 AY331429 AY331430 AY331431 AY331432 AY331432 AY331434 Annals of the Missouri Botanical Garden Campanulaceae Adenophora divaricata Franch. & Sav. Adenophora divaricata Franch. & Sav. Adenophora himalayana Feer Adenophora lobophylla D. Y. Hong Adenophora morrisonensis Hayata Adenophora paniculata Nannf. Adenophora petiolata Pax & Hoffm. Adenophora potaninii Korsh. Adenophora remotiflora (Sieb. & Zucc.) Miq. Adenophora stenanthina (Ledeb.) Kitagawa Adenophora stricta Miq. Adenophora wawreana Zahlbr. Asyneuma japonicum (Miq.) Briq. Azorina vidalii (Wats.) Feer Campanula alliariifolia Willd. Campanula armazica Charadze Campanula arvatica Lag. Campanula barbata L. Campanula bellidifolia Adams Campanula carpatica Jacq. Campanula divaricata Michx. Campanula edulis Forssk. Campanula erinus L. Campanula glomerata L. Campanula grossheimii Kharadze Campanula hawkinsiana Hausskn. & Heldreich Campanula herminii Hoffmans. & Link. Campanula hohenackeri Fisch. & C. A. Mey. DNA accession number and (repository) Voucher information, botanical garden accession number, or reference for published sequences (country of origin when available) 572 Appendix 1. Taxa sequenced for the ITS region of nr DNA, listed alphabetically by genera and species within the Campanulaceae and Lobeliaceae. Institutional abbreviations are: Royal Botanic Gardens Edinburgh (RBGE); Royal Botanic Gardens, Kew (RBGK); Missouri Botanical Garden (MO); Tblisi (TBI); National Campanula Collection, Cambridge (NCC); University of Texas, Austin (UT); Plant Resources Center, University of Texas, Austin (TEX); and University of Edinburgh (EGHB). Accession numbers follow the abbreviations for Botanical Gardens. Material with voucher specimens is followed by collector, collection number, and herbarium acronym. Sources of material for published sequences can be found in the cited publications. Continued. Taxon name and authority Gaskin 466 (UT) Eddie 96051 (UT) Cosner s.n. (UT) Neves 226 (UT) Eddie 96056 (UT) Neves 230 (UT) Gaskin 468 (UT) Eddie 95007 (UT) Eddie 95027 (UT) Eddie s.n. (UT) Neves 229 (UT) Eddie 96092 (UT) Eddie 96089 (UT) Gaskin 57 (UT) Eddie 00004 (UT) Cosner s.n. (UT) Gaskin 458 (UT) Gaskin 462 (UT) Gaskin 314 (UT) Gaskin 302 (UT) Eddie s.n. (UT) Gaskin 417 (MO,UT) Eddie 96050 (UT) Eddie 96048 (UT) Eddie 95022 (UT) Cosner s.n. (UT) Eddie 95023 (UT) AY322022, AY322023, AY322024, AY322025, AY322026, AY322027, AY322028, AY322029, AY322030, AY322031, AY322032, AY322033, AY322034, AY322035, AY322036, AY322037, AY322038, AY322039, AY322040, AY322041, AY322042, AY322043, AY322044, AF134862 AY322045, AY322046, AY322047, AY322048, AF134859 AF136237 AH008217 AF134860 AF134861 AY331435 AY331436 AY331437 AY331438 AY331439 AY331440 AY331441 AY331442 AY331443 AY331444 AY331445 AY331446 AY331447 AY331448 AY331449 AY331450 AY331451 AY331452 AY331453 AY331454 AY331455 AY331456 AY331457 AY331458 AY331459 AY331460 AY331461 573 T. Shulkina 18 (Caucasus, TBI) NCC 96092, Eddie 96051 (EGHB) Lammers, no voucher S. Neves 226 (Portugal, TEX) RBGE 19972042 S. Neves 230 (Spain, TEX) T. Shulkina 58 (Caucasus, TBI) Eddie 95007 (Turkey, TEX) RBGE 1969372, Eddie 95027 (EGHB) RBGE 19860223 (France) S. Neves 229 (Portugal, TEX) NCC 96092, Eddie 96092 (EGHB) NCC, Eddie 96089 (EGHB) T. Shulkina s.n. (Caucasus, Georgia, TBI) Eddie 00004 (USA, Texas, TEX) Lammers 8714 (USA, F) T. Shulkina s.n. (Caucasus, Georgia, TBI) T. Shulkina s.n. (Caucasus, TBI) J. Gaskin 442 (Caucasus, Georgia, TBI) J. Gaskin 158 (Caucasus, Georgia, TBI) NCC, Eddie s.n. (EGHB) J. Gaskin 417 (Caucasus, Georgia, MO) NCC, Eddie 96050 (TEX) Fu et al. (1999) RBGE 19770035 (Spain, Canary Islands) RBGE 19920352 (Nepal) Lammers 8439 (Taiwan, F) RBGE 19870950 Fu et al. (1999) Fu et al. (1999) Fu et al. (1999) Fu et al. (1999) Fu et al. (1999) Genbank accession number (ITS1, ITS2) Eddie et al. Phylogeny of Campanulaceae Campanula kolenatiana C. A. Mey. Campanula lanata Friv. Campanula latifolia L. Campanula lusitanica Loefl. Campanula mirabilis Albov Campanula mollis L. Campanula ossetica Bieb. Campanula peregrina L. Campanula persicifolia L. Campanula petraea L. Campanula primulifolia L. Campanula punctata Lam. Campanula pyramidalis L. Campanula raddeana Trautv. Campanula reverchoni A. Gray Campanula rotundifolia L. Campanula samatica Ker-Gawl. Campanula siegizmundii Fed. Campanula sosnowskyi Charadze Campanula steveni M. Bieb. Campanula thyrsoides L. Campanula tridentata Schreb. Campanulastrum americanum (L.) Small Campanumoea javanica Blume Canarina canariensis (L.) Vatke Codonopsis dicentrifolia W. W. Sm. Codonopsis kawakamii Hayata Codonopsis lanceolata (Sieb & Zucc.) Benth. & Hook.f. Codonopsis modesta Nannf. Codonopsis nervosa Nannf. Codonopsis pilosa Chipp Codonopsis pilosula Nannf. Codonopsis tangshen Oliv. DNA accession number and (repository) Voucher information, botanical garden accession number, or reference for published sequences (country of origin when available) Volume 90, Number 4 2003 Appendix 1. 574 Appendix 1. Continued. Taxon name and authority Eddie 0448 (UT) Cosner s.n. (UT) Eddie 95045 (UT) Eddie 95029 (UT) Eddie 940119 (UT) Eddie 98004F (UT) Eddie 95009 (UT) Cosner s.n. (UT) Eddie 95018 (UT) Haberle 149 (UT) Eddie 95083 (UT) Eddie 95035 (UT) Eddie 49 (UT) Eddie 98 (UT) Eddie 13 (UT) Eddie 97017 (UT) Eddie 95034 (UT) Eddie 95021 (UT) Eddie s.n. (UT) Eddie 95030 (UT) Eddie 96066 (UT) Eddie 95008 (UT) Cosner s.n. (UT) Eddie 96090 (UT) Eddie 96076 (UT) Cosner s.n. (UT) Eddie 760258 (UT) Eddie 750893A (UT) Gaskin 255(UT) Eddie 98008T (UT) Haberle 132 (UT) Eddie 98004W (UT) Hirst & Webster D. 448 (Lesotho, EGHB) Cosner 179 (OS) Eddie 95045 (EGHB) RBGE 19860931 RBGE 19940119 S. L. Jury et al. 17429 (Morocco, TEX) RBGE 19693714 Morin, no voucher RBGE 19872386 (South Korea) R. C Haberle 149 (USA, California, TEX) Eddie 95003 (EGHB, TEX) Eddie 95035 (EGHB) Sales & Hedge 98.49 (Spain, RBGE) Sales & Hedge 98.98 (Spain, RBGE) Sales & Hedge 98.13 (Spain, RBGE) Eddie 97017 (EGHB, TEX) Eddie 95034 (EGHB, TEX) RBGE 198921881 (Nepal) RBGE s.n. Eddie 95030 (EGHB, TEX) Eddie 96066 (Greece, TEX) RBGE 19771648 Lammers 9993 (F) RBGE 19782029 (Spain) Eddie 96076 (EGHB) T. Ayers s. n. (BH) RBGE 19760258 RBGE 19750893 T. Shulkina s.n. (Caucasus, TBI) Eddie 98008T (EGHB) R. C. Haberle 132 (USA, Texas, TEX) Eddie 98004W (Scotland, TEX) Genbank accession number (ITS1, ITS2) AY322049, AY322050, AY322051, AY322052, AY322053, AY322054, AY322055, AY322056, AY322057, AY322058, AY322059, AY322060, AY322061, AY322062, AY322063, AY322064, AY322065, AY322066, AY322068, AY322067, AY322069, AY322070, AY322071, AY322072, AY322073, AY322074, AY322075, AY322076, AY322077, AY322078, AY322079, AY322080, AY331462 AY331463 AY331464 AY331465 AY331466 AY331467 AY331468 AY331469 AY331470 AY331471 AY331472 AY331473 AY331474 AY331475 AY331476 AY331477 AY331478 AY331479 AY331480 AY331481 AY331482 AY331483 AY331484 AY331485 AY331486 AY331487 AY331488 AY331489 AY331490 AY331491 AY331492 AY331493 Annals of the Missouri Botanical Garden Craterocapsa congesta Hilliard & B. L. Burtt Cyananthus lobatus Wall. ex Benth. Diosphaera rumeliana (Hampe) Bornm. Edraianthus graminifolius (L.) A. DC. Edraianthus pumilio (Schultes) A. DC. Feeria angustifolia (Schousb.) Buser Gadellia lactiflora (M. Bieb.) Schulkina Githopsis diffusa A. Gray Hanabusaya asiatica Nakai Heterocodon rariflorum Nutt. Jasione crispa (Pourr.) Samp. Jasione laevis Lam. Jasione maritima (Duby) L. M. Dufour ex Merino Jasione montana L. Jasione sessiliflora Boiss. & Reut. Legousia falcata (Ten.) Fritsch Legousia speculum-veneris (L.) Fisch. Leptocodon gracilis Lem. Michauxia tchihatchewii Fisch. & C. A. Mey. Musschia aurea Dumort. Petromarula pinnata (L.) A. DC. Physoplexis comosa (L.) Schur Phyteuma orbiculare L. Phyteuma spicata L. Platycodon grandiflorus (Jacq.) A. DC. Roella ciliata L. Symphyandra armena (Stev.) A. DC. Symphyandra hofmanni Pant. Symphyandra pendula (Bieb.) A. DC Trachelium caeruleum L. Triodanis leptocarpa (Nutt.) Nieuwl. Wahlenbergia hederacea L. DNA accession number and (repository) Voucher information, botanical garden accession number, or reference for published sequences (country of origin when available) Volume 90, Number 4 2003 Appendix 1. Continued. Taxon name and authority Schultheis (2001) Schultheis (2001) Schultheis (2001) Dotti (1998) Genbank accession number (ITS1, ITS2) AF176900 AF163435 AF163436 AF054938 Eddie et al. Phylogeny of Campanulaceae Lobeliaceae Downingia bacigalupii Weiler Lobelia aberdarica R. E. Fries & T. C. E. Fries Lobelia tupa L. Lobelia tenera Kunth DNA accession number and (repository) Voucher information, botanical garden accession number, or reference for published sequences (country of origin when available) 575

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